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Runx2 transcriptome of prostate cancer cells: insights into invasiveness andbone metastasis

Molecular Cancer 2010, 9:258 doi:10.1186/1476-4598-9-258

Sanjeev K Baniwal (baniwal@usc.edu)Omar Khalid (khalid@usc.edu)Yankel Gabet (gabet@usc.edu)

Ruchir R Shah (ruchir_shah@sra.com)Daniel J Purcell (dpurcell@usc.edu)

Deepak Mav (deepak_mav@sra.com)Alice E Kohn-Gabet (agabet@usc.edu)

Yunfan Shi (yunfansh@usc.edu)Gerhard A Coetzee (coetzee@usc.edu)

Baruch Frenkel (frenkel@usc.edu)

ISSN 1476-4598

Article type Research

Submission date 8 April 2010

Acceptance date 23 September 2010

Publication date 23 September 2010

Article URL http://www.molecular-cancer.com/content/9/1/258

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Molecular Cancer

© 2010 Baniwal et al. , licensee BioMed Central Ltd.This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Runx2 transcriptome of prostate cancer cells: insights into

invasiveness and bone metastasis

Sanjeev K Baniwal1,4,6

, Omar Khalid1,4,6

, Yankel Gabet1,4,6

, Ruchir R Shah7,8

, Daniel J Purcell1,6

,

Deepak Mav7, Alice E Kohn-Gabet

4,6, Yunfan Shi

4,6, Gerhard A Coetzee

3,5,6, Baruch

Frenkel1,2,4,6

1Department of Biochemistry & Molecular Biology, University of Southern California, Los

Angeles, USA

2 Department of Orthopaedic Surgery, University of Southern California, Los Angeles, USA

3 Department of Urology, University of Southern California, Los Angeles, USA

4Institute for Genetic Medicine, Keck School of Medicine at the University of Southern

California, Los Angeles, USA

5Norris Cancer Center, University of Southern California, Los Angeles, USA

6Keck School of Medicine at the University of Southern California, Los Angeles, USA

7SRA International, 2605 Meridian Parkway, Durham, USA

8Sciome LLC, 2 Davis Drive, Research Triangle Park, USA

Email addresses: “Sanjeev K Baniwal” < baniwal@usc.edu>, "Omar Khalid" <khalid@usc.edu>,

"Yankel Gabet" <gabet@usc.edu>, “Ruchir R Shah” < ruchir_shah@sra.com >, "Daniel Purcell"

<dpurcell@usc.edu>, “Deepak Mav” < deepak_mav@sra.com>, "Alice E Kohn-Gabet"

<agabet@usc.edu>, "Samantha Shi" <yunfansh@usc.edu>, "Gerhard Coetzee"

<coetzee@usc.edu>, "Baruch Frenkel" <frenkel@usc.edu>

Corresponding Authors: Baruch Frenkel and Sanjeev K. Baniwal

University of Southern California Keck School of Medicine

Institute for Genetic Medicine

2250 Alcazar Street, CSC 240

Los Angeles, CA 90033

Telephone: (323) 442-1322

FAX: (323) 442-2764

Abstract

Background: Prostate cancer (PCa) cells preferentially metastasize to bone at least in part by

acquiring osteomimetic properties. Runx2, an osteoblast master transcription factor, is aberrantly

expressed in PCa cells, and promotes their metastatic phenotype. The transcriptional programs

regulated by Runx2 have been extensively studied during osteoblastogenesis, where it activates

or represses target genes in a context-dependent manner. However, little is known about the gene

regulatory networks influenced by Runx2 in PCa cells. We therefore investigated genome wide

mRNA expression changes in PCa cells in response to Runx2.

Results: We engineered a C4-2B PCa sub-line called C4-2B/Rx2dox

, in which Doxycycline

(Dox) treatment stimulates Runx2 expression from very low to levels observed in other PCa

cells. Transcriptome profiling using whole genome expression array followed by in silico

analysis indicated that Runx2 upregulated a multitude of genes with prominent cancer associated

functions. They included secreted factors (CSF2, SDF-1), proteolytic enzymes (MMP9, CST7),

cytoskeleton modulators (SDC2, Twinfilin, SH3PXD2A), intracellular signaling molecules

(DUSP1, SPHK1, RASD1) and transcription factors (Sox9, SNAI2, SMAD3) functioning in

epithelium to mesenchyme transition (EMT), tissue invasion, as well as homing and attachment

to bone. Consistent with the gene expression data, induction of Runx2 in C4-2B cells enhanced

their invasiveness. It also promoted cellular quiescence by blocking the G1/S phase transition

during cell cycle progression. Furthermore, the cell cycle block was reversed as Runx2 levels

declined after Dox withdrawal.

Conclusions: The effects of Runx2 in C4-2B/Rx2dox

cells, as well as similar observations made

by employing LNCaP, 22RV1 and PC3 cells, highlight multiple mechanisms by which Runx2

promotes the metastatic phenotype of PCa cells, including tissue invasion, homing to bone and

induction of high bone turnover. Runx2 is therefore an attractive target for the development of

novel diagnostic, prognostic and therapeutic approaches to PCa management. Targeting Runx2

may prove more effective than focusing on its individual downstream genes and pathways.

Introduction

Runx2 together with Runx1 and Runx3 comprise the Runx class of transcription factors, defined

by their highly homologous Runt-related DNA-binding domain. As heterodimers with Cbfß,

Runx proteins bind to cognate DNA elements with the consensus nucleotide sequence 5’-

ACCACA in the promoters/enhancers of their target genes [1]. The three Runx proteins

coordinate proliferation and differentiation of various cell types [2]. Runx1 is important for

hematopoiesis [3, 4]; Runx2 is pivotal in osteogenesis [1, 5, 6]; and Runx3 is critical for

neurogenesis [7], thymopoiesis [4], and maintenance of the gastric epithelium [4, 8]. While

promoting specific cellular phenotypes, Runx proteins have evolved to inhibit cell proliferation.

Runx3 is a bona fide tumor suppressor [9] as down-regulation of its promoter by

hypermethylation contributes to the development of gastric cancer [10, 11]. Ablation of Runx1

activity leads to leukemia [12] and disruption of Runx2 results in deregulated cell proliferation

and immortalization [13-17]. Paradoxically, Runx2 is also implicated in carcinogenesis. In a

mouse screen for c-Myc-collaborating oncogenes, MLV-induced leukemia occurred most

frequently when the provirus integrated into the Runx2 locus resulting in its ectopic expression

[18]. It was suggested that Runx2 initially provides the cells with a survival advantage, and its

anti-mitogenic activity is counteracted by the CD2-Myc transgene present in the mouse model

used for this screen [2, 19]. Therefore, Runx2-mediated tumorigenesis likely requires additional

loss of check-point genes such as Trp53 or improper regulation of an oncogene such as c-Myc

[19].

Runx2 has been extensively studied in the context of osteoblastogenesis from mesenchymal

progenitors, where as a master regulator it stimulates the expression of various bone matrix

components such as osteocalcin and bone sialoprotein (BSP) [20]. Runx2-/-

mice die soon after

birth due to the lack of differentiated osteoblasts and thus a mineralized skeleton [1, 5, 6]. Runx2

haploinsufficiency in humans causes the rare skeletal disorder Cleidocranial Dysplasia [21]. In

search for hints to explain the high predilection of prostate and breast cancer to metastasize to

bone, investigators have noticed ectopic expression of Runx2 and some of its target genes in

biopsies from advanced tumors and their derivative cell lines [22-26]. In a mouse model of PCa,

conditional deletion of Pten in prostate epithelial cells resulted in the development of tumors

with progressive increase in Runx2 expression [27]. Among the osteomimetic properties of

prostate and breast cancer cells are expression of the Runx2 target genes MMP9 [28], BSP [29]

and VEGFA [30], as well as induction of mineralization [25].

In addition to promoting osteoblast differentiation, Runx2 drives the expression of

osteoclastogenic signals, both in osteoblasts [31, 32] and in the PC3 bone metastasis-derived PCa

cell line [22]. PC3 cells robustly express Runx2 [33], and its silencing decreased their

osteoclastogenic property in vitro and their growth within the bone microenvironment in vivo

[22]. Runx2 also promotes metastatic aspects not necessarily related to bone. Invasion of PC3

cells through Matrigel™, a basement membrane-like preparation, decreased after Runx2

silencing [22], and its ectopic expression in mammary epithelial cells increased their

proliferation and disrupted their normal acinar organization [34]. An oncogenic role for Runx2

has also been suggested in tumors that do not exhibit high predilection to bone, including

pancreatic ductal adenocarcinoma [35] and thyroid papillary carcinoma [36].

Whereas Runx2 is being increasingly recognized as a pro-metastatic factor, little is known about

the underlying transcriptional programs. To establish gene regulatory networks downstream of

Runx2 in aggressive PCa, we analyzed gene expression in response to Runx2 in the C4-2B PCa

cell line. These cells are castration-resistant derivatives of the androgen-dependent LNCaP cells,

and serve as a model for the aggressive stage of bone metastatic PCa [37, 38]. Although C4-2B

cells express Runx2 at levels higher than LNCaP cells [25], these levels are far lower than those

observed in PC3 cells or osteoblasts [22]. We therefore engineered a C4-2B sub-line that

allowed us to profile gene expression after induction of Runx2 with Doxycycline to levels seen

in PC3 cells. Remarkably, the most significant changes were the up-regulation of genes

implicated in cancer progression and cellular movement, and the down-regulation of genes

involved in cell cycle progression. Consistent with these changes in gene expression, Runx2

enhanced PCa cell invasiveness and inhibited their proliferation.

Results and Discussion

Establishment of C4-2B PCa cells with conditional Runx2 expression

To establish a C4-2B cell line that conditionally expresses Runx2, we employed the recently

described lentivirus-based pSLIK vector system, which allows tight Doxycycline (Dox)-

inducible, RNA PolII-mediated transcription of a gene of interest [39]. C4-2B cells were

transduced with Flag-tagged Runx2-encoding lentiviruses (Figure 1A), resulting in the C4-

2B/Rx2dox

subline. As control, we established the C4-2B/Rx2-Mdox

subline, where Dox treatment

induced expression of the transcriptionally inactive Flag-Runx2-M (Figure 1A) [40]. Western

blot analysis with anti-Flag antibodies confirmed roughly equal expression levels of the wild

type and mutant Runx2 proteins, which were strictly and dose-dependently regulated by Dox

(Figure 1B). RT-qPCR analysis revealed that the Dox treatment increased Runx2 mRNA by ~20-

fold compared to its endogenous levels, and that the induced level was comparable to that

observed in the PC3high

sub-line (Figure 1C). Western analysis using anti-Runx2 antibodies

indicated that the level of endogenous Runx2 protein was negligible in untreated C4-2B cells,

and that Dox induced expression of the exogenous Runx2 to the levels normally found in

osteoblasts (Figure 1D) [41].

The transcriptional activity of Dox-induced Runx2 was initially assessed using luciferase

reporter assay (Figure 1E). In the reporter plasmid 6XOSE2-Luc, luciferase expression was

controlled by six copies of the osteoblast-specific element 2 (OSE2) from the Runx2-regulated

osteocalcin (OC) gene promoter [42]. In the absence of Dox, 6XOSE2-luc activity was

indistinguishable from the background luciferase activity observed without any cell extract,

suggesting lack of endogenous Runx2 activity (Figure 1E). The luciferase reporter was strongly

stimulated by WT but not by the mutant form of Runx2 (Figure 1E). As shown in Figure 1F,

Runx2 also stimulated transcription of its endogenous target genes Bone Sialoprotein (BSP) [29]

and Matrix Metalloprotease-9 (MMP9) [28]. These genes were not stimulated in the Dox-treated

C4-2B/Rx2-Mdox

cells (data not shown). Interestingly, Runx2 did not significantly enhance the

expression of OC (Figure 1F) and Alkaline Phophatase (ALP; data not shown), although these

genes are strongly stimulated by Runx2 in osteoblasts [1, 43]. This observation reflects cell type-

dependent Runx2-mediated transcriptional control, and is consistent with the results of Yeung et

al. [33], who demonstrated that in PC3 cells the OC promoter is responsive to the transcription

factors AP-1 and SP1, but not Runx2. To identify Runx2-regulated genes and pathways in

advanced PCa cells in an unbiased manner, we subjected C4-2B/Rx2dox

cells to global gene

expression profiling.

Runx2-regulated global gene expression and in silico assessment of associated pathways

C4-2B/Rx2dox

cells were subjected to microarray gene expression analysis after one- and two-

days of treatment with either Dox or vehicle in biological quadruplicates (a total of 16 samples).

Of 24,526 probes represented in the microarray, 532 genes showed ≥2-fold increased expression

and 378 genes showed ≥2-fold decreased expression with high statistical significance (p<0.008)

on either day of treatment (see additional file 1). RT-qPCR analysis of 50 representative genes

conformed to the microarray data (Table 1 and additional file 2).

An unsupervised hierarchical analysis of these 910 up- and down-regulated genes resulted in a

clear separation between the Dox-treated and control samples (Figure 2). The variation among

the biological quadruplicates was small, indicating the overall robustness of the methodology

utilized. Gene clusters showing changes in expression pattern with respect to time and Dox

treatment were clearly discernable. In general, changes observed on day 1 of treatment were

maintained or intensified by day 2.

We next employed the Ingenuity Pathway Analysis (IPA™) platform to indentify disease

pathways, as well as molecular and cellular functions associated with Runx2-regulated genes.

The analysis suggested ‘cancer’ as the disease most significantly associated with both the up-

and the down-regulated gene groups (Table 2). A total of 248 genes, half from each group, had

highly significant cancer related function (see additional file 3). Additionally, the up-regulated

genes were strongly associated with genetic disorders, inflammatory responses, and

gastrointestinal diseases (Table 2A). Among the most significant molecular and cellular

functions, cellular movement, cell death, cellular growth, and proliferation were associated with

the up-regulated genes, whereas cell cycle, cell death, cellular assembly and DNA replication

functions were associated with the down-regulated genes (Table 2B).

Runx2-modulated genes are involved in tumor metastasis.

Promotion of tissue invasion, metastasis and cytoskeleton dynamics: The major functions

reported for the up-regulated genes belonged to cancer progression (Table 2). Importantly, these

genes encode transcriptional regulators, cytoskeletal components, signaling molecules and

peptidases, which have been implicated in tumor metastasis (Table 1). The transcription factors

Sox9 and SNAI2, and the extracellular-matrix (ECM) protein LCN2, all major regulators of

epithelial-to-mesenchymal transition (EMT) [44, 45], were up-regulated by ~4-fold after one day

and by >6-fold after two days of Runx2 induction, and their upregulation was confirmed by RT-

qPCR (Table 1). However, the functional significance of these EMT markers requires further

investigation in light of the unexpected increase in E-cadherin mRNA (see additional file 4).

Runx2 also enhanced the expression levels of multiple transcripts encoding matrix modifying

peptidases (Table 1). These included MMP9, a known Runx2 target in BCa cells [28] and

aspartyl proteases with fibronectin degrading activities such as Prolactin Induced Protein (PIP)

and Pepsinogen (PGC) [46, 47]. The latter two showed a rapid ~10-fold increase within 24 hours

(Table 1) and PIP exhibited the highest change (79-fold) in response to Runx2 on day 2 (Table 1

and additional file 2). PIP protein in the C4-2B/Rx2dox

culture supernatant was below detectable

levels under control conditions, but was readily detected after induction of Runx2 (Figure 3). We

also found increased transcript levels for Cystatin 7 (CST7), S100A4 and SMAD3, with a mild

~3-fold increase on day 1, but a robust >20-fold increase on day 2 (Table 1). These genes

function as metastasis promoters [48, 49]. Interestingly, S100A4 and SMAD3 physically interact

to potentiate cancer cell invasiveness [50].

Runx2 also up-regulated genes involved in cellular movement and cytoskeleton remodeling

(Table 1). SH3PXD2A, which was up-regulated by 7-fold on day 2, is a scaffold protein

involved in the formation of invadopedia [51, 52], which are matrix digesting, actin rich, short

lived protrusions observed in osteoclasts and cancer cells [53]. Runx2 up-regulated by >9-fold

the transcripts for Nav2, a scaffold protein crucial for actin cytoskeleton remodeling [53]. Other

genes that were up-regulated by >3-fold, with known roles in actin cytoskeleton dynamics

included ESPN, which interacts with the Src-homology 3 (SH3) adaptor proteins to regulate

cytoskeletal actin functions [54]; MAP1B, known to maintain cytoskeletal integrity [55];

LIMA1, which cross-links actin monomers [56]; and PTK9L (a.k.a. twinfiln), which sequesters

ADP-actin monomers in the cytoplasm and delivers them to sites of rapid actin-filament

assembly [57].

Metastasis to bone and modification of the bone microenvironment: The expression of SDF-1

and its receptor CXCR7 was enhanced by >5-fold based on the microarray analysis and by >20-

fold based on the RT-qPCR results (Table 1). Runx2-induced SDF-1 protein was also detectable

in the culture supernatant (Figure 3). SDF-1 signaling is critical for homing of hematopoietic

cells to the bone marrow space and their survival in this environment [58-65]. Within one day,

Runx2 also increased by 10-fold the mRNA for BSP (Figure 1F), whose abundant expression by

bone metastatic tumor cells facilitates their attachment to the bone matrix [66-69]. Once settled

in the bone microenvironment, the metastatic cells secrete regulatory molecules that stimulate

bone turnover [70]. Remarkably, Runx2 enhanced the expression of the osteoclastogenic

cytokine CSF2 by >50-fold within 48 hours (Table 1). This presumably occurred by direct

binding of Runx2 to the CSF2 promoter [71]. Runx2-mediated induction of CSF2 in PCa cells

likely contributes to the increased bone turnover in bone metastatic sites, similar to the role of

this cytokine in breast cancer bone metastasis [72]. CSF2 production by tumor cells may also

contribute to accumulation of macrophages, inflammatory T cells, and cytokines [73, 74] that

exacerbate morbidity and mortality [75]. Two additional Runx2-up-regulated genes associated

with osteoclast function are SPHK1, a kinase responsible for the production of sphingosine 1

phosphate (S1P), and S1P receptor 3 (S1P3) a.k.a. EDG3 (Table 1). Production of S1P in the

bone microenvironment promotes bone resorption by chemotactically attracting osteoclast

precursors [76]. The SPHK1/S1P/S1P3 axis plays additional roles in cancer progression,

including cell growth, migration, angiogenesis, and resistance to chemotherapy [77-79]. Notably,

Runx2 was the only gene differentially up-regulated in chemotherapy-resistant versus -sensitive

osteosarcoma tumors [80]. Adding to this, Runx2 also repressed the expression of GDF-15, an

osteoclastogenesis inhibitor [81] (Table 1). This repression was mild on day 1 (2-fold), but by

day 2 GDF-15 was the most repressed gene in response to Runx2 (8-fold; Table 1 and additional

file 2). Thus, Runx2-mediated alterations in gene expression may contribute to both the

predilection of PCa to bone and the subsequent pathological increase in bone turnover, which

further fuels growth of the metastatic tumors.

Angiogenesis: Runx2 has been implicated in promoting angiogenesis by stimulating VEGFA

expression during bone development [30] as well as during tumorigenesis [82]. In C4-2B/Rx2dox

cells, Runx2 increased VEGFA mRNA by 4-fold (Table 1) and the presence of VEGF in the cell

culture supernatant was detectable only after Dox treatment (Figure 3). Additionally, our study

revealed a 32-fold upregulation of the VEGFA co-receptor Syndecan-2 (SDC2; Table 1). SDC2,

which is also a Runx2 target in osteoprogenitor cells [83], is a member of the heparan sulfate

proteoglycans family, and is also implicated in cell adhesion and communication [84-86].

Interestingly, VEGFA can functionally synergize with SDF-1 to promote neoangiogenesis in

vivo [62]. Our microarray analysis also revealed Runx2-mediated induction of the angiogenic

EDN-2 gene (Table 1). Endothelins and VEGFA are secreted by PCa cells to stimulate

angiogenesis as well as differentiation of neighboring osteoblasts in the bone microenvironment

[72, 87-91].

Runx2 increases the invasion potential of C4-2B cells in vitro

Because Runx2 enhanced the expression of multiple extracellular enzymes involved in ECM

degradation (Table 1), we initially tested by in-gel zymography the presence of proteases in the

supernatant of Dox-treated C4-2B/Rx2dox

cultures. The results demonstrated that Runx2 induced

several gelatin-degrading proteins, in particular one with a molecular weight of ~140 kDa, the

identity of which remains to be determined (Figure 4A). We further investigated whether Runx2

stimulates invasion of C4-2B/Rx2dox

cells through Matrigel™, a tissue basement membrane-like

preparation containing laminin, type IV collagen, heparan sulfate proteoglycans and entactin. For

convenient and accurate assessment of cells that successfully invade through the Matrigel™

membrane, we transduced the C4-2B/Rx2dox

cells with a lentivirus constitutively expressing

luciferase. Parallel trans-wells that do not contain Matrigel™ were employed as migration

controls. Cells were incubated in the respective chambers in the presence or absence of Dox, and

the relative migration or invasion capacity was assessed. Runx2 expression led to a 2.3-fold

decrease in cell migration, but a 4.3-fold increase in invasion through Matrigel™, i.e., a ~10-fold

increase in invasiveness after adjustment for the reduced cell migration (Figure 4B). The

increased invasiveness was further confirmed in an independent experiment by histological

staining (Figure 4C). In parallel experiments, expression of Runx2-M in C4-2B cells showed no

significant effects on either migration or invasion (data not shown). Thus, expression of

transcriptionally active Runx2 is sufficient to enhance the tissue invasion potential of C4-2B

cells.

Runx2 induces cellular quiescence by reversibly inhibiting G1/S cell cycle transition

The IPA™ analysis of Runx2 down-regulated genes indicated their strong association with cell

cycle and proliferation-related functions (Table 2B). Many of these down-regulated genes, as

well as several up-regulated genes, shed light on the well-established anti-proliferative activity of

Runx2 [2, 15-17, 19]. Most striking was the >19- and >8-fold up-regulation of RASD1 and

DUSP1, respectively (Table 1). RASD1 belongs to the Ras superfamily of G-proteins, and its

expression in breast cancer suppressed cell growth [92]. DUSP1, a.k.a. MAP kinase phosphatase

1 (MKP1), is a dual specificity (Thr/Tyr) protein phophatase with anti-proliferative properties

[93]. Among the most important cell cycle regulatory genes inhibited by Runx2 was c-Myc, with

a ~3-fold decrease at the mRNA level (Table 1) and a corresponding significant decrease at the

protein level (Figure 3). In line with the down-regulation of c-Myc, the mRNA encoding its cell

cycle promoting targets E2F2 and CDK2 were also down-regulated (Table 1). CDK2 protein was

decreased below detectability (Figure 3). To further characterize effects of Runx2 on PCa cell

proliferation, we first validated by RT-qPCR the changes in the transcript levels of RASD1,

DUSP1, c-Myc and E2F2 in the day-2 samples (Figure 5A). Next, we tested the effect of Runx2

on C4-2B cell proliferation by performing MTT assays every 48 hours after Dox-mediated

Runx2 induction. Runx2 significantly restrained cell proliferation (Figure 5B). By contrast, the

transcriptionally-inactive Runx2-M did not affect proliferation (Figure 5C). Thus, Runx2

restrains PCa cell proliferation via its transcriptional activation property.

To delineate the anti-proliferative effect of Runx2, we tested its influence on apoptosis and cell

cycle progression. Apoptosis was measured using succinyl-AMC, a fluorogenic Caspase-3

substrate. Expression of Runx2, but not Runx2-M, resulted in a transient 40% increase in

apoptosis on day 4 (Figure 5D), but this could not account for the dramatic inhibition of cell

proliferation (Figure 5B). Instead, fluorescence activated cell-sorting (FACS) analysis revealed a

2.2-fold decrease in the fraction of cells in the S/G2/M phases of the cell cycle within 24 hours

of Dox treatment, and this effect persisted on day 2 (Figure 5E). Notably, the cell cycle

inhibition preceded any change in apoptosis, indicating that Runx2 restrained C4-2B cell

proliferation by inhibiting the G1/S phase transition of the cell cycle.

The prometastatic but anti-mitogenic properties of Runx2 in PCa cells suggest that it may

initially facilitate metastasis, after which it must be degraded or antagonized (e.g. by co-

repressors) to allow cell proliferation and tumor growth. Therefore, we examined if the anti-

mitogenic effect of Runx2 was reversible by withdrawing Dox from cultures after 48 hours of

treatment (Figure 5F). Dox withdrawal led to substantial clearance of Runx2 within two days,

and undetectable Runx2 levels after four days (Figure 5G). This resulted in resumption of cell

cycle progression (Figure 5H) and cell proliferation (Figure 5I). Thus, the Runx2-regulated gene

networks induce reversible cellular quiescence by blocking the G1/S phase transition of the cell

cycle.

Generality and Network modeling of Runx2-regulated genes with cancer-related functions

Our study is the first to provide genome wide analysis of Runx2-regulated genes in PCa cells.

Although we focused on the bone metastasis-derived C4-2B cells, similar responses to Runx2

were observed in the parental lymph node-derived LNCaP cells [38] (see additional file 5) as

well as in the unrelated bone metastatic 22RV1 PCa cells (see additional file 6) [94].

Furthermore, in PC3high

and PC3low

cells with high and low levels of Runx2, respectively, the

expression of six randomly selected Runx2 up-regulated genes from the present study correlated

with Runx2 expression (see additional file 7). Together, these results suggest that our

observations are relevant to various stages of PCa progression. Among the 910 genes that Runx2

up- or down-regulated by ≥2 fold, IPA™ identified 248 genes related to cancer with high

statistical significance (p<0.003; Table 2 and additional file 3). The IPA™ analysis, as well as

survey of literature on gene expression profiling in osteoblasts and fibroblasts, further suggested

that the Runx2-regulated gene network in PCa (Figure 6) bears little resemblance to its targets in

mesenchymal cells [43, 83, 95-97]. In fact, only five of the cancer-related genes in our study

have been previously reported as Runx2 targets (MMP13, CEBPA, VEGF, SMAD3, and

SMAD6) and only six others are Runx1 and/or Runx3 targets (SKP2, CSF2, IL3, ACHE,

IGFBP3, and HIPK2; Figure 6). The remaining 234 genes are therefore novel Runx2-regulated

genes related to cancer in general and to metastasis in particular. The extensive Runx2-regulated

cancer-related gene network highlights Runx2 as a viable target for the diagnosis, prognosis and

treatment of PCa.

Conclusions

Runx2, traditionally known for its master regulatory roles in the chondro-osteoblast lineage, is

emerging as a prometastatic transcription factor [22, 29, 34, 98-101]. The Runx2 transcriptome

in C4-2B cells documents gene networks that control multiple aspects of metastasis. Potentially

contributing to local invasion and dissemination are the genes known to function in EMT,

motility and ECM degradation. Additionally, the prometastatic function of Runx2 likely involves

its target genes SDF-1, CXCR7 and BSP, which promote homing and attachment to bone. We

also discovered Runx2 targets such as CSF2 and SPHK1, osteoclast activators that likely

contribute to the most important alteration that occurs in the bone microenvironment in response

to PCa metastasis, namely enhanced bone turnover. During this process, bone matrix

components such as TGFß, BMPs and calcium ions are released and further fuel tumor growth

and bone microenvironment modifications [102-105]. The regulation of SPHK1 by Runx2

probably potentiates additional aspects of the cancer phenotype, including angiogenesis (a

function assisted by the Runx2 targets VEGFA and EDN2) and drug resistance [77, 79].

The anti-mitogenic activity of Runx2 is consistent with the slow growth of PCa tumors, and may

contribute to drug resistance. We imagine that future anti-Runx2 drugs will be administered

along with traditional chemotherapy to eliminate cells that regain proliferative capacity.

Interestingly, Runx2 physically and functionally interacts with the receptors for androgens and

estrogens [99, 101]. Since these receptor proteins are targeted by many drugs for prostate and

breast cancer, it is important to investigate their effects on Runx2-regulated transcription.

Moreover, development of selective estrogen and androgen receptor modulators may benefit

from consideration of their effects on Runx2 and expression of its target genes reported in the

present study.

Methods

Cell culture reagents and antibodies: C4-2B cells were obtained from ViroMed Laboratories

(Minneapolis, MN). LNCaP and 22RV1 cells were from ATCC (Rockville, MD, USA). PC3

cells were also obtained from ATCC, but propagated for several years in either our laboratory

(PC3low

) or that of USC’s Dr. Pradip Roy-Burman (PC3high

). The cells were maintained in

RPMI-1640 medium supplemented with 10% Tet System Approved FBS from Clontech, CA,

USA. Hygromycin B was purchased from Invitrogen, Carlsbad, CA, USA and added to the

growth medium at 50 µg/ml. Doxycycline from Calibiochem, La Jolla, CA, USA was used at

0.25 ug/ml unless otherwise stated, and an equal volume of distilled water was used as vehicle

control. Mouse ANTI-FLAG® M2 monoclonal antibody for was purchased from Sigma, St

Louis, MO, USA. Mouse anti-Runx2 was from Invitrogen, Carlsbad, CA, USA. The anti-PIP

antibody (ab 62363) was purchased from abcam Inc., Cambridge, MA; and anti-GAPDH (V-18),

anti-TGFß-RIII (Sc 28975), and anti-SDF-1 (Sc-28876) antibodies were purchased from Santa

Cruz Biotechnology Inc, Santa Cruz, CA, USA. The mouse monoclonal anti-Myc and anti-

VEGFA antibodies were developed and kindly provided by USC’s Dr. Prakash S. Gill and Dr.

Young Hong, respectively. The mouse monoclonal anti-Tubulin antibody, developed by Dr.

Charles Walsh, was obtained from the Developmental Studies Hybridoma Bank under the

auspices of the NICHD and The University of Iowa, Department of Biological Sciences, Iowa

City, USA.

Plasmid construction: The cDNA encoding mouse Runx2 (MASN isoform, type-2), which is

97% identical to human type-2 Runx2 [1], was amplified using pcDNA3.0-Runx2 as template

and the Flag epitope was inserted during the PCR amplification (see primers in additional file 8).

The Flag-Runx2 cDNA was initially cloned into the SpeI/MfeI-digested lentiviral entry vector

pEN_TmiRc3 (ATCC® catalog: MBA-248), and the resulting plasmid was recombined using

Gateway® LR Clonase® II enzyme mix (Invitrogen, Carlsbad, CA, USA) with the pSLIK

(single lentivector for inducible knockdown) destination vector carrying a hygromycin resistance

gene (ATCC® catalog: MBA-237). The entry and destination vectors were kindly provided by

USC’s Dr. Elizabeth Lowler (Childrens Hospital Los Angeles). The DNA-binding mutant

Runx2-M was constructed by site directed mutagenesis of two arginine residues at positions 265

and 268 (GRSGRGK) known to contact DNA in the crystal structure [40]. Lentiviral plasmid for

constitutive Luciferase expression pCCL-c-MNDU3c-Luc-PGK-eGFP was kindly provided by

USC’s Dr. Michael Kahn at the Zilkha Neurogenetic Institute.

Lentivirus production and infection: For packaging, the lentiviral expression plasmids were

cotransfected by the calcium chloride method into HEK293T cells along with helper plasmids

pMD.G1 and pCMV R8.91[106, 107]. Culture media containing viral particles were harvested

after 48–72 hours and used for transduction of C4-2B cells in the presence of 8 µg/ml Polybrene

(Millipore Corp., MA, USA). After infection with the lentiviruses, the transduced cells were

selected with 50 µg/ml of Hygromycin.

Transient transfection and Luciferase assays: Transient transfection and Luciferase assays were

performed essentially as described by Khalid et al. [99]. Briefly, 25,000 cells were plated in a 24-

well plate 48 hours prior to transfection using Invitrogen’s Lipofectamine™ LTX reagent

according to manufacturer’s instructions. Cells were harvested and subjected to luciferase assay

using Luciferase Assay system from Promega, Madison, WI, USA.

High throughput gene expression measurement and analysis: Gene expression profiling was

performed using the BeadChipTM

platform (Illumina) and chip reference 8, version 3 for humans,

which contains 24,526 gene probes and 664 negative control probes. Details of the raw data

processing and analysis are provided in the additional file 9. Briefly, after background correction,

the normalized expression intensities for all probes were subjected to a two-way analysis of

variance (ANOVA) and the resulting p-values were adjusted for multiple testing using the

Benjimini-Hochberg method [108]. The differentially expressed probes were further investigated

using the Ingenuity Pathways Analysis package (IPA™; www.ingenuity.com) to identify

biological functions and disease categories that are significantly enriched among the

differentially expressed genes. Right-tailed Fisher’s exact test as implemented in the IPA

software was used to calculate a p-value for the probability of each network to be enriched for

Runx2-regulated genes due to chance alone. The microarray data has been deposited to the GEO

database with the accession code GSE24261.

Preparation of conditioned medium and gelatin zymography: C4-2B/Rx2dox

cells were cultured

in 10 cm culture dishes to 80% confluence, washed 3 times with RPMI-1640 and treated with

Dox in 10 mL of RPMI-1640 without FBS for 24 hrs. For zymography the 25 µl of the

conditioned media from Dox treated or control cells were analyzed by 8% acrylamide gels

containing 0.1% w/v gelatin [109] After electrophoresis, the gel was washed with 2.5% Triton

X-100 and incubated in the developing buffer (500mM Tris-HCl, pH 7.8, 2M NaCl, 50 mM

CaCl2) overnight to induce gelatin lysis. Gel was stained by Coomasie blue-250. For western blot

analysis of conditioned media, 250 µl of the supernatant was TCA-precipitated and subjected to

reducing SDS-PAGE analysis using standard procedures.

In vitro invasion and migration assays: Invasion through Matrigel™ was assessed by incubating

20,000 luciferase-expressing cells at the top of 24 well BD BioCoat™ Growth Factor Reduced

Matrigel™ chamber (BD Biosciences, MA, USA). Migration was assessed using BD BioCoat™

Control Inserts. Cells that migrated or invaded to the bottom compartment were visualized by

Diff-Quick™ staining kit (Baxter, MN, USA) or subjected to luciferase assays for quantification.

Invasion index was defined as the percentage of cells that invaded through Matrigel™ over those

that migrated under the same conditions but without the Matrigel™.

Proliferation, cell cycle and apoptosis: Cultures on different days were subjected to MTT assay

(Sigma, St Louis, MO) to measure viable cells in culture. For cell cycle analysis, 2×105

to 1×106

cells were harvested, washed twice with PBS (1 mL) at room temperature and stored in absolute

ethanol (4 mL) for at least 24 hours. Pelleted cells were rehydrated in 5 mL PBS for 15 minutes,

followed by staining with 1 mL of a propidium iodide (PI, Calibiochem, La jolla, CA, USA)

solution containing 3 µM PI in incubation buffer (100 mM Tris, pH 7.4, 150 mM NaCl, 1mM

CaCl2, 0.5 mM MgCl2, 0.1% Nonidet® P-40). The cell suspension (1 mL) was subjected to

fluorescence-activated cell sorting (FACScaliber, Becton Dickinson, MA, USA) and each cell

was assigned to the G1, S, G2 or M phase of the cell cycle based on the PI intensity and using

the Multicycle v3.0 software (Phoenix Flow Systems, San Diego, CA, USA). To assess

apoptosis, cells were lysed in caspase assay buffer containing 50 mM HEPES (pH 7.5), 100 mM

NaCl, 2 mM EDTA, 0.1% CHAPS, 10% sucrose, and 5 mM DTT. Aliquots of crude cell lysate

(50 µg protein) were incubated with the caspase-3 substrate Ac-DEVD-AMC

(EMD/Calbiochem, La Jolla, CA, USA) at 37°C for 30 min and the caspase-3 activity was

quantified by flow fluorimetry with excitation at 380 nm and emission

at 440 nm using

Victor3V™ from PerkinElmer, Shelton, CT, USA.

Western blot analysis: Between 1×105 and 2×10

5 cells were washed once with PBS and lysed

with 200 µL of incubation buffer [100 mM Tris (pH 7.4), 500 mM NaCl, 1 mM CaCl2, 0.5 mM

MgCl2, 0.1% Nonidet®

P-40] supplemented with Complete™ protease inhibitor mix (Roche

Diagnostics, Indianapolis, IN, USA). Aliquots of 40 µg cell lysate were mixed with an equal

volume of Laemmli buffer and proteins were resolved by 10% SDS-PAGE, transferred to

Amersham Hybond™-P PVDF (GE Healthcare, Piscataway, NJ, USA) membranes, and

visualized using respective antibodies and the Western Lightning™ Plus-ECL kit (PerkinElmer

Inc, Waltham, MA, USA) followed by exposure to X-ray film (ISCBioExpress®, Kaysville, UT,

USA).

RT-qPCR: Total RNA was isolated using Aurum Total RNA kit (Bio-Rad Laboratories Inc.,

Hercules, CA, USA) following the manufacturer’s recommendations and 1 µg was reverse

transcribed using the Superscript III kit (Invitrogen, CA, USA). The cDNA was subjected to real-

time PCR amplification using iQ SYBR Green Supermix and a Opticon™2 real time PCR

machine from Bio-Rad, Hercules, CA. The sequences of primers for amplification of the cDNA

of interest and the control GAPDH are listed in the additional file 8.

GEO accession code for the microarray data: GSE24261

Competing interests

The authors declare no competing interests

Authors' contributions

Conceived and designed the experiments: SKB and BF. Performed the experiments: SKB, OK,

YG, AEG, SS and DJP. Bioinformatics analysis: RRS and DM. Analyzed data and wrote the

paper: SKB, GAC and BF. All authors have read and approved the final manuscript.

Acknowledgements

We thank Dr. Joseph Hacia and Dr. Nyam-Osor Chimge at the University of Southern California

for their critical comments during the preparation of the manuscript. We also thank Dr. Gerard

Karsenty (Columbia University, New York, NY) for the 6XOSE2-luciferase reporter and

pCDNA3.1-Runx2 (mouse) plasmids. The microarray analysis was performed at the Southern

California Genotyping Consortium at UCLA (http://scgc.genetics.ucla.edu/) under the direction

of Mr. Joseph DeYoung. Further analysis of the gene expression profiles was performed with

help from Dr. Yibu Chen at the USC Norris Medical Library. We thank Dr. Shinwu Jeong and

Dr. Gabriel M Gordon for their help with the zymography assay. We also thank Dr. Pradip Roy-

Burman and Helty Adisetiyo for kindly providing the PC3high

cell line. The anti-TGFß-RIII

antibody was kindly provided by Dr. Yang Chai at Center for Craniofacial Molecular Biology;

and anti-SDF-1 antibody was kindly provided by Dr. Henry Sucov at the University of Southern

California. This work was funded by NIH grants R01 CA 109147 and R01 CA 136924. SKB was

supported by a postdoctoral Innovative Chapter Research Award; and YG by a Meyer Young

Investigator Fellowship, both from the Arthritis Foundation Southern California Chapter. BF

holds the J. Harold and Edna L. LaBriola Chair in Genetic Orthopedic Research at USC.

Reference:

1. Ducy P, Zhang R, Geoffroy V, Ridall AL, Karsenty G: Osf2/Cbfa1: a transcriptional

activator of osteoblast differentiation. Cell 1997, 89:747-754.

2. Cameron ER, Neil JC: The Runx genes: lineage-specific oncogenes and tumor

suppressors. Oncogene 2004, 23:4308-4314.

3. Lo Coco F, Pisegna S, Diverio D: The AML1 gene: a transcription factor involved in

the pathogenesis of myeloid and lymphoid leukemias. Haematologica 1997, 82:364-

370.

4. Woolf E, Xiao C, Fainaru O, Lotem J, Rosen D, Negreanu V, Bernstein Y, Goldenberg

D, Brenner O, Berke G, et al: Runx3 and Runx1 are required for CD8 T cell

development during thymopoiesis. Proc Natl Acad Sci U S A 2003, 100:7731-7736.

5. Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, Deguchi K, Shimizu Y, Bronson

RT, Gao YH, Inada M, et al: Targeted disruption of Cbfa1 results in a complete lack

of bone formation owing to maturational arrest of osteoblasts. Cell 1997, 89:755-764.

6. Otto F, Thornell AP, Crompton T, Denzel A, Gilmour KC, Rosewell IR, Stamp GW,

Beddington RS, Mundlos S, Olsen BR, et al: Cbfa1, a candidate gene for cleidocranial

dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell 1997, 89:765-771.

7. Levanon D, Bettoun D, Harris-Cerruti C, Woolf E, Negreanu V, Eilam R, Bernstein Y,

Goldenberg D, Xiao C, Fliegauf M, et al: The Runx3 transcription factor regulates

development and survival of TrkC dorsal root ganglia neurons. Embo J 2002,

21:3454-3463.

8. Ito Y: Oncogenic potential of the RUNX gene family: 'overview'. Oncogene 2004,

23:4198-4208.

9. Bae SC, Choi JK: Tumor suppressor activity of RUNX3. Oncogene 2004, 23:4336-

4340.

10. Levanon D, Groner Y: Structure and regulated expression of mammalian RUNX

genes. Oncogene 2004, 23:4211-4219.

11. Oshimo Y, Oue N, Mitani Y, Nakayama H, Kitadai Y, Yoshida K, Ito Y, Chayama K,

Yasui W: Frequent loss of RUNX3 expression by promoter hypermethylation in

gastric carcinoma. Pathobiology 2004, 71:137-143.

12. Rossetti S, Van Unen L, Touw IP, Hoogeveen AT, Sacchi N: Myeloid maturation block

by AML1-MTG16 is associated with Csf1r epigenetic downregulation. Oncogene

2005, 24:5325-5332.

13. Kilbey A, Blyth K, Wotton S, Terry A, Jenkins A, Bell M, Hanlon L, Cameron ER, Neil

JC: Runx2 disruption promotes immortalization and confers resistance to oncogene-

induced senescence in primary murine fibroblasts. Cancer Res 2007, 67:11263-

11271.

14. Zaidi SK, Pande S, Pratap J, Gaur T, Grigoriu S, Ali SA, Stein JL, Lian JB, van Wijnen

AJ, Stein GS: Runx2 deficiency and defective subnuclear targeting bypass

senescence to promote immortalization and tumorigenic potential. Proc Natl Acad

Sci U S A 2007, 104:19861-19866.

15. Galindo M, Pratap J, Young DW, Hovhannisyan H, Im HJ, Choi JY, Lian JB, Stein JL,

Stein GS, van Wijnen AJ: The bone-specific expression of Runx2 oscillates during the

cell cycle to support a G1-related antiproliferative function in osteoblasts. J Biol

Chem 2005, 280:20274-20285.

16. Pratap J, Galindo M, Zaidi SK, Vradii D, Bhat BM, Robinson JA, Choi JY, Komori T,

Stein JL, Lian JB, et al: Cell growth regulatory role of Runx2 during proliferative

expansion of preosteoblasts. Cancer Res 2003, 63:5357-5362.

17. Thomas DM, Johnson SA, Sims NA, Trivett MK, Slavin JL, Rubin BP, Waring P,

McArthur GA, Walkley CR, Holloway AJ, et al: Terminal osteoblast differentiation,

mediated by runx2 and p27KIP1, is disrupted in osteosarcoma. J Cell Biol 2004,

167:925-934.

18. Blyth K, Terry A, Mackay N, Vaillant F, Bell M, Cameron ER, Neil JC, Stewart M:

Runx2: a novel oncogenic effector revealed by in vivo complementation and

retroviral tagging. Oncogene 2001, 20:295-302.

19. Blyth K, Cameron ER, Neil JC: The RUNX genes: gain or loss of function in cancer.

Nat Rev Cancer 2005, 5:376-387.

20. Komori T: Regulation of osteoblast differentiation by transcription factors. J Cell

Biochem 2006, 99:1233-1239.

21. Otto F, Kanegane H, Mundlos S: Mutations in the RUNX2 gene in patients with

cleidocranial dysplasia. Hum Mutat 2002, 19:209-216.

22. Akech J, Wixted JJ, Bedard K, van der Deen M, Hussain S, Guise TA, van Wijnen AJ,

Stein JL, Languino LR, Altieri DC, et al: Runx2 association with progression of

prostate cancer in patients: mechanisms mediating bone osteolysis and osteoblastic

metastatic lesions. Oncogene 2009:811-821.

23. Koeneman KS, Yeung F, Chung LW: Osteomimetic properties of prostate cancer

cells: a hypothesis supporting the predilection of prostate cancer metastasis and

growth in the bone environment. Prostate 1999, 39:246-261.

24. Chua CW, Chiu YT, Yuen HF, Chan KW, Man K, Wang X, Ling MT, Wong YC:

Suppression of androgen-independent prostate cancer cell aggressiveness by

FTY720: validating Runx2 as a potential antimetastatic drug screening platform. Clin Cancer Res 2009, 15:4322-4335.

25. Lin DL, Tarnowski CP, Zhang J, Dai J, Rohn E, Patel AH, Morris MD, Keller ET: Bone

metastatic LNCaP-derivative C4-2B prostate cancer cell line mineralizes in vitro. Prostate 2001, 47:212-221.

26. Selvamurugan N, Kwok S, Partridge NC: Smad3 interacts with JunB and

Cbfa1/Runx2 for transforming growth factor-beta1-stimulated collagenase-3

expression in human breast cancer cells. J Biol Chem 2004, 279:27764-27773.

27. Lim M, Zhong C, Yang S, Bell AM, Cohen MB, Roy-Burman P: Runx2 regulates

survivin expression in prostate cancer cells. Lab Invest, 90:222-233.

28. Pratap J, Javed A, Languino LR, van Wijnen AJ, Stein JL, Stein GS, Lian JB: The

Runx2 osteogenic transcription factor regulates matrix metalloproteinase 9 in bone

metastatic cancer cells and controls cell invasion. Mol Cell Biol 2005, 25:8581-8591.

29. Barnes GL, Javed A, Waller SM, Kamal MH, Hebert KE, Hassan MQ, Bellahcene A,

Van Wijnen AJ, Young MF, Lian JB, et al: Osteoblast-related transcription factors

Runx2 (Cbfa1/AML3) and MSX2 mediate the expression of bone sialoprotein in

human metastatic breast cancer cells. Cancer Res 2003, 63:2631-2637.

30. Zelzer E, Glotzer DJ, Hartmann C, Thomas D, Fukai N, Soker S, Olsen BR: Tissue

specific regulation of VEGF expression during bone development requires

Cbfa1/Runx2. Mech Dev 2001, 106:97-106.

31. Geoffroy V, Kneissel M, Fournier B, Boyde A, Matthias P: High bone resorption in

adult aging transgenic mice overexpressing cbfa1/runx2 in cells of the osteoblastic

lineage. Mol Cell Biol 2002, 22:6222-6233.

32. Maruyama Z, Yoshida CA, Furuichi T, Amizuka N, Ito M, Fukuyama R, Miyazaki T,

Kitaura H, Nakamura K, Fujita T, et al: Runx2 determines bone maturity and

turnover rate in postnatal bone development and is involved in bone loss in estrogen

deficiency. Dev Dyn 2007, 236:1876-1890.

33. Yeung F, Law WK, Yeh CH, Westendorf JJ, Zhang Y, Wang R, Kao C, Chung LW:

Regulation of human osteocalcin promoter in hormone-independent human

prostate cancer cells. J Biol Chem 2002, 277:2468-2476.

34. Pratap J, Imbalzano KM, Underwood JM, Cohet N, Gokul K, Akech J, van Wijnen AJ,

Stein JL, Imbalzano AN, Nickerson JA, et al: Ectopic runx2 expression in mammary

epithelial cells disrupts formation of normal acini structure: implications for breast

cancer progression. Cancer Res 2009, 69:6807-6814.

35. Kayed H, Jiang X, Keleg S, Jesnowski R, Giese T, Berger MR, Esposito I, Lohr M,

Friess H, Kleeff J: Regulation and functional role of the Runt-related transcription

factor-2 in pancreatic cancer. Br J Cancer 2007, 97:1106-1115.

36. Endo T, Ohta K, Kobayashi T: Expression and function of Cbfa-1/Runx2 in thyroid

papillary carcinoma cells. J Clin Endocrinol Metab 2008, 93:2409-2412.

37. Horoszewicz JS, Leong SS, Kawinski E, Karr JP, Rosenthal H, Chu TM, Mirand EA,

Murphy GP: LNCaP model of human prostatic carcinoma. Cancer Res 1983,

43:1809-1818.

38. Wu HC, Hsieh JT, Gleave ME, Brown NM, Pathak S, Chung LW: Derivation of

androgen-independent human LNCaP prostatic cancer cell sublines: role of bone

stromal cells. Int J Cancer 1994, 57:406-412.

39. Shin KJ, Wall EA, Zavzavadjian JR, Santat LA, Liu J, Hwang JI, Rebres R, Roach T,

Seaman W, Simon MI, Fraser ID: A single lentiviral vector platform for microRNA-

based conditional RNA interference and coordinated transgene expression. Proc

Natl Acad Sci U S A 2006, 103:13759-13764.

40. Tahirov TH, Inoue-Bungo T, Morii H, Fujikawa A, Sasaki M, Kimura K, Shiina M, Sato

K, Kumasaka T, Yamamoto M, et al: Structural analyses of DNA recognition by the

AML1/Runx-1 Runt domain and its allosteric control by CBFbeta. Cell 2001,

104:755-767.

41. Sudhakar S, Li Y, Katz MS, Elango N: Translational regulation is a control point in

RUNX2/Cbfa1 gene expression. Biochem Biophys Res Commun 2001, 289:616-622.

42. Ducy P, Karsenty G: Two distinct osteoblast-specific cis-acting elements control

expression of a mouse osteocalcin gene. Mol Cell Biol 1995, 15:1858-1869.

43. Vaes BL, Ducy P, Sijbers AM, Hendriks JM, van Someren EP, de Jong NG, van den

Heuvel ER, Olijve W, van Zoelen EJ, Dechering KJ: Microarray analysis on Runx2-

deficient mouse embryos reveals novel Runx2 functions and target genes during

intramembranous and endochondral bone formation. Bone 2006, 39:724-738.

44. Cheung M, Chaboissier MC, Mynett A, Hirst E, Schedl A, Briscoe J: The

transcriptional control of trunk neural crest induction, survival, and delamination. Dev Cell 2005, 8:179-192.

45. Yang J, Bielenberg DR, Rodig SJ, Doiron R, Clifton MC, Kung AL, Strong RK,

Zurakowski D, Moses MA: Lipocalin 2 promotes breast cancer progression. Proc Natl

Acad Sci U S A 2009, 106:3913-3918.

46. Caputo E, Manco G, Mandrich L, Guardiola J: A novel aspartyl proteinase from

apocrine epithelia and breast tumors. J Biol Chem 2000, 275:7935-7941.

47. Chiang L, Contreras L, Chiang J, Ward PH: Human prostatic gastricsinogen: the

precursor of seminal fluid acid proteinase. Arch Biochem Biophys 1981, 210:14-20.

48. Morita M, Yoshiuchi N, Arakawa H, Nishimura S: CMAP: a novel cystatin-like gene

involved in liver metastasis. Cancer Res 1999, 59:151-158.

49. Sherbet GV: Metastasis promoter S100A4 is a potentially valuable molecular target

for cancer therapy. Cancer Lett 2009, 280:15-30.

50. Matsuura I, Lai CY, Chiang KN: Functional interaction between Smad3 and S100A4

(metastatin-1) for TGF-beta-mediated cancer cell invasiveness. Biochem J 2010,

426:327-335.

51. Artym VV, Zhang Y, Seillier-Moiseiwitsch F, Yamada KM, Mueller SC: Dynamic

interactions of cortactin and membrane type 1 matrix metalloproteinase at

invadopodia: defining the stages of invadopodia formation and function. Cancer Res

2006, 66:3034-3043.

52. Larsen M, Artym VV, Green JA, Yamada KM: The matrix reorganized: extracellular

matrix remodeling and integrin signaling. Curr Opin Cell Biol 2006, 18:463-471.

53. Schmidt KL, Marcus-Gueret N, Adeleye A, Webber J, Baillie D, Stringham EG: The cell

migration molecule UNC-53/NAV2 is linked to the ARP2/3 complex by ABI-1. Development 2009, 136:563-574.

54. Sekerkova G, Loomis PA, Changyaleket B, Zheng L, Eytan R, Chen B, Mugnaini E,

Bartles JR: Novel espin actin-bundling proteins are localized to Purkinje cell

dendritic spines and bind the Src homology 3 adapter protein insulin receptor

substrate p53. J Neurosci 2003, 23:1310-1319.

55. Riederer BM: Microtubule-associated protein 1B, a growth-associated and

phosphorylated scaffold protein. Brain Res Bull 2007, 71:541-558.

56. Maul RS, Song Y, Amann KJ, Gerbin SC, Pollard TD, Chang DD: EPLIN regulates

actin dynamics by cross-linking and stabilizing filaments. J Cell Biol 2003, 160:399-

407.

57. Paavilainen VO, Bertling E, Falck S, Lappalainen P: Regulation of cytoskeletal

dynamics by actin-monomer-binding proteins. Trends Cell Biol 2004, 14:386-394.

58. Levoye A, Balabanian K, Baleux F, Bachelerie F, Lagane B: CXCR7 heterodimerizes

with CXCR4 and regulates CXCL12-mediated G protein signaling. Blood 2009,

113:6085-6093.

59. Mazzinghi B, Ronconi E, Lazzeri E, Sagrinati C, Ballerini L, Angelotti ML, Parente E,

Mancina R, Netti GS, Becherucci F, et al: Essential but differential role for CXCR4

and CXCR7 in the therapeutic homing of human renal progenitor cells. J Exp Med

2008, 205:479-490.

60. Kryczek I, Wei S, Keller E, Liu R, Zou W: Stroma-derived factor (SDF-1/CXCL12)

and human tumor pathogenesis. Am J Physiol Cell Physiol 2007, 292:C987-995.

61. Singh S, Singh UP, Grizzle WE, Lillard JW, Jr.: CXCL12-CXCR4 interactions

modulate prostate cancer cell migration, metalloproteinase expression and invasion. Lab Invest 2004, 84:1666-1676.

62. Kryczek I, Lange A, Mottram P, Alvarez X, Cheng P, Hogan M, Moons L, Wei S, Zou L,

Machelon V, et al: CXCL12 and vascular endothelial growth factor synergistically

induce neoangiogenesis in human ovarian cancers. Cancer Res 2005, 65:465-472.

63. Taichman RS, Cooper C, Keller ET, Pienta KJ, Taichman NS, McCauley LK: Use of the

stromal cell-derived factor-1/CXCR4 pathway in prostate cancer metastasis to bone. Cancer Res 2002, 62:1832-1837.

64. Ara T, Tokoyoda K, Sugiyama T, Egawa T, Kawabata K, Nagasawa T: Long-term

hematopoietic stem cells require stromal cell-derived factor-1 for colonizing bone

marrow during ontogeny. Immunity 2003, 19:257-267.

65. Peled A, Petit I, Kollet O, Magid M, Ponomaryov T, Byk T, Nagler A, Ben-Hur H, Many

A, Shultz L, et al: Dependence of human stem cell engraftment and repopulation of

NOD/SCID mice on CXCR4. Science 1999, 283:845-848.

66. Waltregny D, Bellahcene A, Van Riet I, Fisher LW, Young M, Fernandez P, Dewe W, de

Leval J, Castronovo V: Prognostic value of bone sialoprotein expression in clinically

localized human prostate cancer. J Natl Cancer Inst 1998, 90:1000-1008.

67. Waltregny D, Bellahcene A, de Leval X, Florkin B, Weidle U, Castronovo V: Increased

expression of bone sialoprotein in bone metastases compared with visceral

metastases in human breast and prostate cancers. J Bone Miner Res 2000, 15:834-

843.

68. Zhang JH, Tang J, Wang J, Ma W, Zheng W, Yoneda T, Chen J: Over-expression of

bone sialoprotein enhances bone metastasis of human breast cancer cells in a mouse

model. Int J Oncol 2003, 23:1043-1048.

69. Jung K, Lein M, Stephan C, Von Hosslin K, Semjonow A, Sinha P, Loening SA, Schnorr

D: Comparison of 10 serum bone turnover markers in prostate carcinoma patients

with bone metastatic spread: diagnostic and prognostic implications. Int J Cancer

2004, 111:783-791.

70. Keller ET, Zhang J, Cooper CR, Smith PC, McCauley LK, Pienta KJ, Taichman RS:

Prostate carcinoma skeletal metastases: cross-talk between tumor and bone. Cancer

Metastasis Rev 2001, 20:333-349.

71. Liu H, Holm M, Xie XQ, Wolf-Watz M, Grundstrom T: AML1/Runx1 recruits

calcineurin to regulate granulocyte macrophage colony-stimulating factor by Ets1

activation. J Biol Chem 2004, 279:29398-29408.

72. Park BK, Zhang H, Zeng Q, Dai J, Keller ET, Giordano T, Gu K, Shah V, Pei L, Zarbo

RJ, et al: NF-kappaB in breast cancer cells promotes osteolytic bone metastasis by

inducing osteoclastogenesis via GM-CSF. Nat Med 2007, 13:62-69.

73. Grivennikov SI, Greten FR, Karin M: Immunity, inflammation, and cancer. Cell 2010,

140:883-899.

74. Dranoff G: Cytokines in cancer pathogenesis and cancer therapy. Nat Rev Cancer

2004, 4:11-22.

75. Greten FR, Eckmann L, Greten TF, Park JM, Li ZW, Egan LJ, Kagnoff MF, Karin M:

IKKbeta links inflammation and tumorigenesis in a mouse model of colitis-

associated cancer. Cell 2004, 118:285-296.

76. Ishii M, Egen JG, Klauschen F, Meier-Schellersheim M, Saeki Y, Vacher J, Proia RL,

Germain RN: Sphingosine-1-phosphate mobilizes osteoclast precursors and regulates

bone homeostasis. Nature 2009, 458:524-528.

77. Shida D, Takabe K, Kapitonov D, Milstien S, Spiegel S: Targeting SphK1 as a new

strategy against cancer. Curr Drug Targets 2008, 9:662-673.

78. Visentin B, Vekich JA, Sibbald BJ, Cavalli AL, Moreno KM, Matteo RG, Garland WA,

Lu Y, Yu S, Hall HS, et al: Validation of an anti-sphingosine-1-phosphate antibody as

a potential therapeutic in reducing growth, invasion, and angiogenesis in multiple

tumor lineages. Cancer Cell 2006, 9:225-238.

79. Pchejetski D, Golzio M, Bonhoure E, Calvet C, Doumerc N, Garcia V, Mazerolles C,

Rischmann P, Teissie J, Malavaud B, Cuvillier O: Sphingosine kinase-1 as a

chemotherapy sensor in prostate adenocarcinoma cell and mouse models. Cancer

Res 2005, 65:11667-11675.

80. Sadikovic B, Thorner P, Chilton-Macneill S, Martin JW, Cervigne NK, Squire J,

Zielenska M: Expression analysis of genes associated with human osteosarcoma

tumors shows correlation of RUNX2 overexpression with poor response to

chemotherapy. BMC Cancer, 10:202.

81. Vanhara P, Lincova E, Kozubik A, Jurdic P, Soucek K, Smarda J:

Growth/differentiation factor-15 inhibits differentiation into osteoclasts--a novel

factor involved in control of osteoclast differentiation. Differentiation 2009, 78:213-

222.

82. Pratap J, Lian JB, Javed A, Barnes GL, van Wijnen AJ, Stein JL, Stein GS: Regulatory

roles of Runx2 in metastatic tumor and cancer cell interactions with bone. Cancer

Metastasis Rev 2006, 25:589-600.

83. Teplyuk NM, Haupt LM, Ling L, Dombrowski C, Mun FK, Nathan SS, Lian JB, Stein

JL, Stein GS, Cool SM, van Wijnen AJ: The osteogenic transcription factor Runx2

regulates components of the fibroblast growth factor/proteoglycan signaling axis in

osteoblasts. J Cell Biochem 2009, 107:144-154.

84. Essner JJ, Chen E, Ekker SC: Syndecan-2. Int J Biochem Cell Biol 2006, 38:152-156.

85. Park H, Kim Y, Lim Y, Han I, Oh ES: Syndecan-2 mediates adhesion and

proliferation of colon carcinoma cells. J Biol Chem 2002, 277:29730-29736.

86. Granes F, Urena JM, Rocamora N, Vilaro S: Ezrin links syndecan-2 to the

cytoskeleton. J Cell Sci 2000, 113 ( Pt 7):1267-1276.

87. Nelson JB, Hedican SP, George DJ, Reddi AH, Piantadosi S, Eisenberger MA, Simons

JW: Identification of endothelin-1 in the pathophysiology of metastatic

adenocarcinoma of the prostate. Nat Med 1995, 1:944-949.

88. Dai J, Kitagawa Y, Zhang J, Yao Z, Mizokami A, Cheng S, Nor J, McCauley LK,

Taichman RS, Keller ET: Vascular endothelial growth factor contributes to the

prostate cancer-induced osteoblast differentiation mediated by bone morphogenetic

protein. Cancer Res 2004, 64:994-999.

89. Street J, Bao M, deGuzman L, Bunting S, Peale FV, Jr., Ferrara N, Steinmetz H, Hoeffel

J, Cleland JL, Daugherty A, et al: Vascular endothelial growth factor stimulates bone

repair by promoting angiogenesis and bone turnover. Proc Natl Acad Sci U S A 2002,

99:9656-9661.

90. Cross MJ, Claesson-Welsh L: FGF and VEGF function in angiogenesis: signalling

pathways, biological responses and therapeutic inhibition. Trends Pharmacol Sci

2001, 22:201-207.

91. Ferrara N, Gerber HP, LeCouter J: The biology of VEGF and its receptors. Nat Med

2003, 9:669-676.

92. Vaidyanathan G, Cismowski MJ, Wang G, Vincent TS, Brown KD, Lanier SM: The

Ras-related protein AGS1/RASD1 suppresses cell growth. Oncogene 2004, 23:5858-

5863.

93. Horsch K, de Wet H, Schuurmans MM, Allie-Reid F, Cato AC, Cunningham J, Burrin

JM, Hough FS, Hulley PA: Mitogen-activated protein kinase phosphatase 1/dual

specificity phosphatase 1 mediates glucocorticoid inhibition of osteoblast

proliferation. Mol Endocrinol 2007, 21:2929-2940.

94. Sramkoski RM, Pretlow TG, 2nd, Giaconia JM, Pretlow TP, Schwartz S, Sy MS,

Marengo SR, Rhim JS, Zhang D, Jacobberger JW: A new human prostate carcinoma

cell line, 22Rv1. In Vitro Cell Dev Biol Anim 1999, 35:403-409.

95. Hecht J, Seitz V, Urban M, Wagner F, Robinson PN, Stiege A, Dieterich C, Kornak U,

Wilkening U, Brieske N, et al: Detection of novel skeletogenesis target genes by

comprehensive analysis of a Runx2(-/-) mouse model. Gene Expr Patterns 2007,

7:102-112.

96. Young DW, Hassan MQ, Yang XQ, Galindo M, Javed A, Zaidi SK, Furcinitti P,

Lapointe D, Montecino M, Lian JB, et al: Mitotic retention of gene expression patterns

by the cell fate-determining transcription factor Runx2. Proc Natl Acad Sci U S A

2007, 104:3189-3194.

97. Wotton S, Terry A, Kilbey A, Jenkins A, Herzyk P, Cameron E, Neil JC: Gene array

analysis reveals a common Runx transcriptional programme controlling cell

adhesion and survival. Oncogene 2008, 27:5856-5866.

98. Shore P: A role for Runx2 in normal mammary gland and breast cancer bone

metastasis. J Cell Biochem 2005, 96:484-489.

99. Khalid O, Baniwal SK, Purcell DJ, Leclerc N, Gabet Y, Stallcup MR, Coetzee GA,

Frenkel B: Modulation of Runx2 activity by estrogen receptor-alpha: implications

for osteoporosis and breast cancer. Endocrinology 2008, 149:5984-5995.

100. Javed A, Barnes GL, Pratap J, Antkowiak T, Gerstenfeld LC, van Wijnen AJ, Stein JL,

Lian JB, Stein GS: Impaired intranuclear trafficking of Runx2 (AML3/CBFA1)

transcription factors in breast cancer cells inhibits osteolysis in vivo. Proc Natl Acad

Sci U S A 2005, 102:1454-1459.

101. Baniwal SK, Khalid O, Sir D, Buchanan G, Coetzee GA, Frenkel B: Repression of

Runx2 by androgen receptor (AR) in osteoblasts and prostate cancer cells: AR binds

Runx2 and abrogates its recruitment to DNA. Mol Endocrinol 2009, 23:1203-1214.

102. Morrissey C, Vessella RL: The role of tumor microenvironment in prostate cancer

bone metastasis. J Cell Biochem 2007, 101:873-886.

103. Roodman GD: Mechanisms of bone metastasis. N Engl J Med 2004, 350:1655-1664.

104. Mundy GR: Metastasis to bone: causes, consequences and therapeutic opportunities.

Nat Rev Cancer 2002, 2:584-593.

105. Logothetis CJ, Lin SH: Osteoblasts in prostate cancer metastasis to bone. Nat Rev

Cancer 2005, 5:21-28.

106. Phillips JE, Garcia AJ: Retroviral-mediated gene therapy for the differentiation of

primary cells into a mineralizing osteoblastic phenotype. Methods Mol Biol 2008,

433:333-354.

107. Kim JH, Yang CK, Heo K, Roeder RG, An W, Stallcup MR: CCAR1, a key regulator

of mediator complex recruitment to nuclear receptor transcription complexes. Mol

Cell 2008, 31:510-519.

108. Benjamini Y, Hochberg Y: Controlling the false discovery rate: a practical and

powerful approach to multiple testing. Journal of the Royal Statistical Society Series B

1995, 57:289-300.

109. Toth M, Fridman R: Assessment of Gelatinases (MMP-2 and MMP-9 by Gelatin

Zymography Metastasis Research Protocols 2008, 57:163-174.

110. Thiery JP: Epithelial-mesenchymal transitions in tumour progression. Nat Rev

Cancer 2002, 2:442-454.

111. Dang CV, O'Donnell KA, Zeller KI, Nguyen T, Osthus RC, Li F: The c-Myc target

gene network. Semin Cancer Biol 2006, 16:253-264.

112. Guccione E, Martinato F, Finocchiaro G, Luzi L, Tizzoni L, Dall' Olio V, Zardo G, Nervi

C, Bernard L, Amati B: Myc-binding-site recognition in the human genome is

determined by chromatin context. Nat Cell Biol 2006, 8:764-770.

113. Burns JM, Summers BC, Wang Y, Melikian A, Berahovich R, Miao Z, Penfold ME,

Sunshine MJ, Littman DR, Kuo CJ, et al: A novel chemokine receptor for SDF-1 and

I-TAC involved in cell survival, cell adhesion, and tumor development. J Exp Med

2006, 203:2201-2213.

114. Bellou S, Hink MA, Bagli E, Panopoulou E, Bastiaens PI, Murphy C, Fotsis T: VEGF

autoregulates its proliferative and migratory ERK1/2 and p38 cascades by

enhancing the expression of DUSP1 and DUSP5 phosphatases in endothelial cells. Am J Physiol Cell Physiol 2009, 297:C1477-1489.

115. Liu F, Verin AD, Wang P, Day R, Wersto RP, Chrest FJ, English DK, Garcia JG:

Differential regulation of sphingosine-1-phosphate- and VEGF-induced endothelial

cell chemotaxis. Involvement of G(ialpha2)-linked Rho kinase activity. Am J Respir

Cell Mol Biol 2001, 24:711-719.

116. Liu Y, Wada R, Yamashita T, Mi Y, Deng CX, Hobson JP, Rosenfeldt HM, Nava VE,

Chae SS, Lee MJ, et al: Edg-1, the G protein-coupled receptor for sphingosine-1-

phosphate, is essential for vascular maturation. J Clin Invest 2000, 106:951-961.

117. Donaudy F, Zheng L, Ficarella R, Ballana E, Carella M, Melchionda S, Estivill X,

Bartles JR, Gasparini P: Espin gene (ESPN) mutations associated with autosomal

dominant hearing loss cause defects in microvillar elongation or organisation. J Med

Genet 2006, 43:157-161.

118. Bartles JR, Wierda A, Zheng L: Identification and characterization of espin, an actin-

binding protein localized to the F-actin-rich junctional plaques of Sertoli cell

ectoplasmic specializations. J Cell Sci 1996, 109 ( Pt 6):1229-1239.

119. Gu C, Rodriguez ER, Reimert DV, Shu T, Fritzsch B, Richards LJ, Kolodkin AL, Ginty

DD: Neuropilin-1 conveys semaphorin and VEGF signaling during neural and

cardiovascular development. Dev Cell 2003, 5:45-57.

120. Singh R, Bandyopadhyay D: MUC1: a target molecule for cancer therapy. Cancer

Biol Ther 2007, 6:481-486.

121. Tanner SL, Franzen R, Jaffe H, Quarles RH: Evidence for expression of some

microtubule-associated protein 1B in neurons as a plasma membrane glycoprotein. J Neurochem 2000, 75:553-562.

122. Stylli SS, Stacey TT, Verhagen AM, Xu SS, Pass I, Courtneidge SA, Lock P: Nck

adaptor proteins link Tks5 to invadopodia actin regulation and ECM degradation. J

Cell Sci 2009, 122:2727-2740.

123. Donalies M, Cramer M, Ringwald M, Starzinski-Powitz A: Expression of M-cadherin,

a member of the cadherin multigene family, correlates with differentiation of

skeletal muscle cells. Proc Natl Acad Sci U S A 1991, 88:8024-8028.

124. Hu SI, Carozza M, Klein M, Nantermet P, Luk D, Crowl RM: Human HtrA, an

evolutionarily conserved serine protease identified as a differentially expressed gene

product in osteoarthritic cartilage. J Biol Chem 1998, 273:34406-34412.

Figure Legends

Figure 1. Establishment of C4-2B/Rx2dox

sub-line with conditional Runx2 expression. A,

Schematic diagram of the pSLIK-based lentiviral vector. Initially developed for expression of

shRNAs [39] the pSLIK vector was used in the present study to express Runx2. The Hygromycin

resistance marker (Hyg) and the Dox-dependent activator protein rtTA3 are constitutively

expressed under the control of the Ubi-c promoter. Upon treatment with Dox, rtTA binds to its

tetracycline responsive elements (TRE) and drives expression of the inserted cDNA (Flag-

Runx2). The Runx2 block diagram depicts its glutamine/alanine-rich QA domain, the DNA-

binding Runt domain, and the proline/serine/threonine-rich PST domain. Arrowhead indicates the

position of the R265D and R268D mutations in Runx2-M, which eliminate Runx2’s DNA

binding function. B, Whole cell extracts, prepared from C4-2B/Rx2dox

and C4-2B/Rx2-Mdox

cells

treated with the indicated concentrations of Dox, were subjected to western blot analysis using

anti-Flag antibodies. C, Total RNA was extracted from C4-2B/Rx2dox

cells treated with Dox or

vehicle, as well as from PC3high

cells, and the mRNA levels of Runx2 (and GAPDH as control)

were measured by RT-qPCR. D, Whole cell extracts were prepared from C4-2B/Rx2dox

cells

treated with Dox as indicated and from ROS 17.8/2 osteoblastic cells [41], and subjected to

western blot analysis using anti-Runx2 antibodies. The same blot was re-probed with anti-

Tubulin antibodies as loading control. E, C4-2B/Rx2dox

and C4-2B/Rx2-Mdox

cells were

transiently transfected with the 6XOSE2-luciferase reporter plasmid and subjected to luciferase

assay. Dotted line represents the background luciferase activity with no cell extract. F, C4-

2B/Rx2dox

cells were treated with Dox and levels of the indicated transcripts were measured by

RT-qPCR and corrected for that of GAPDH. In Figure 1, bars represent Mean±SEM (n=3) from

a representative experiment, which was repeated at least three times with similar results.

Abbreviations used: Dox, Doxycycline; Veh, vehicle; BSP, Bone Sialoprotein; MMP9, Matrix

Metalloprotein-9; OC, Osteocalcin.

Figure 2. Unsupervised hierarchical clustering of differentially expressed genes. Genes that

Runx2 up- or down-regulated by ≥2-fold on either day 1 or day 2 (total: 910 genes) with p<0.008

were subjected to pearson’s centered correlation matrix. Heatmap represents intensity values

relative to the median intensity across all 16 samples per probe after background subtraction and

normalization.

Figure 3. Runx2-regulated protein expression. C4-2B/Rx2dox

cells were treated with Dox or

vehicle control, and proteins extracted as whole cell lysate (upper panel) or the supernatant

(bottom panel) were subjected to western blot analysis using the indicated antibodies.

Figure 4. Runx2 enhances the invasiveness of C4-2B cells. A) Zymography of supernatants

from C4-2B/Rx2dox

cells treated with either Dox or vehicle. Negatively-stained bands represent

the activity of gelatin-degrading proteases. B) C4-2B/Rx2dox

/Luc cells were incubated in the top

chambers (inserts) of the Matrigel™ invasion system. Cell passage through inserts with and

without Matrigel™ represent invasion and migration, respectively. Identical number of cells was

seeded in the indicated inserts for 24 hours and the cells that appeared on the bottom side

(outside) of the inserts were solubilized in lysis buffer and subjected to luciferase assay. C) The

invasion of cells through the Matrigel™ membrane was assessed by staining with Diff-Quick™

solution.

Figure 5. Runx2 inhibits the G1/S phase transition of the cell cycle. A, RT-qPCR analysis of

the indicated genes in C4-2B/Rx2dox

cells treated with Dox (filled bars) or vehicle (open bars) for

48 hours. B and C, MTT-based cell proliferation assays of C4-2B/Rx2dox

and C4-2B/Rx2-Mdox

cells treated with Dox or vehicle as depicted for the indicated time periods. D, Relative apoptosis

based on caspase 3 activity in whole cell extracts prepared from C4-2B/Rx2dox

and C4-2B/Rx2-

Mdox

cells after treatment with Dox or vehicle for the indicated time periods. E, FACS-based cell

cycle analysis of propidium iodide-stained C4-2B/Rx2dox

cells treated with Dox for the indicated

time periods. F-I, Schematic description of Dox treatment and its subsequent withdrawal from

the cell cultures (F). Samples were harvested at the indicated times (arrows) and subjected to

analysis of Runx2 levels by western blotting (G), cell cycle profiling by FACS analysis (H), and

cell proliferation by MTT assays (I). Abbreviations: RASD1, RAS, Dexamethasone-induced 1;

DUSP, Dual Specificity Phosphatase.

Figure 6. Runx2-regulated cancer-related gene network. The 119 cancer-related genes

showing a significant ≥2-fold response to Runx2 on day 1 were subjected to the pathway

analysis tool from Ingenuity Systems (IPA™). Direct relationships with Runx2 are shown as

thick lines and interactions with its paralogs Runx1 and Runx3 are shown as dashed lines. Red

and blue fonts mark up- and down-regulated genes, respectively.

Additional files

Additional file 1

Title: ANOVA analysis of the microarray data.

Description: List of all probes with the microarray gene expression data in log scale (base 2)

along with the Fold change, P-values and gene annotations.

Additional file 2

Title: Runx2-regulated genes involved in cellular metabolism.

Description: List of genes with functions in cellular metabolism, their fold changes, and known

functions.

Additional file 3

Title: Runx2 -regulated genes involved in cancer.

Description: List of 256 genes with established roles in cancer.

Additional file 4

Title: E-Cadherin expression in C4-2B/Rx2dox

cells upon Runx2 expression.

Description: Western blot and RT-qPCR analysis of C4-2B/Rx2dox

cells in response to Runx2

expression.

Additional file 5

Title: Generation and characterization of LNCaP/Rx2dox

cells.

Description: RT-PCR to detect Runx2 transcript in PC3, C4-2B, and LNCaP cells. Proliferation

of the LNCaP/Rx2dox

cells by using MTT, and RT-qPCR analysis of Runx2-regulated genes.

Additional file 6

Title: Generation and characterization of 22RV1/Rx2dox

cells.

Description: Western blot analysis of Dox-induced Runx2, and MTT based proliferation analysis

of 22RV1/Rx2dox

cells in response to Runx2 expression.

Additional file 7

Title: Expression of PGC, CST7, S100A4, SDF-1, CSF2, and DUSP1 in two PC3 sub-lines with

different Runx2 levels.

Description: RT-qPCR analysis using PC3high

and PC3low

cells to examine the expression of

Runx2-regulated genes.

Additional file 8

Title: List of primers used in the present study to PCR-amplify the indicated gene sequences.

Description: Nucleotide sequence of primers.

Additional file 9

Title: Bioinformatics analysis of the microarray data.

Description: Detailed description of microarray quality control and data processing (such as

quality control, normalization and box/density plots); and methodology employed for the

identification of differentially expressed probes.

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1903

E

DG

3

Sp

hin

gosi

ne-

1-p

hosp

hat

e re

cep

tor

3

4.8

E-1

0

3

7

G

PC

R,

enh

ance

s tu

mor

gro

wth

, ce

ll m

igra

tion

, an

d a

ngio

gen

esis

[7

7-7

9,

115

, 116

]

8877

S

PH

K1

S

phin

gosi

ne

kin

ase

1

7.5

E-0

9

3

12

3

6

Kin

ase

for

the

pro

duct

ion

of

S1

P,

enhan

ces

tum

or

gro

wth

, ce

ll m

igra

tion

, an

d

angio

gen

esis

, P

Ca

cell

su

rviv

al a

gai

nst

ch

emoth

erap

y,

ost

eocl

ast

chem

ota

xis

[77

-79,

115

, 116

]

7049

T

GF

BR

3

Tra

nsf

orm

ing g

row

th f

acto

r, β

rec

epto

r II

I 7

.1E

-09

3

4

1

2

Neg

ativ

e re

gu

lati

on

of

TG

Fβ

rec

epto

r si

gnal

ing p

ath

way

49

21

D

DR

2

Dis

coid

in d

om

ain

rec

epto

r ty

rosi

ne

kin

ase

2 2

.9E

-05

2

5

Coll

agen

bin

din

g, ce

ll p

roli

fera

tion

1017

C

DK

2

Cycl

in-d

epen

den

t kin

ase

2

1.3

E-0

3

-2

-3

-4

Kin

ase,

mit

oti

c ce

ll c

ycl

e, c

ell

pro

life

rati

on

5503

8

CD

CA

4

Cel

l d

ivis

ion c

ycl

e as

soci

ated

4

7.2

E-0

8

-2

-4

-4

Rep

ress

or

of

E2

F-d

epen

den

t tr

ansc

ripti

onal

act

ivat

ion

and

cel

l p

roli

fera

tion

5997

R

GS

2

Reg

ula

tor

of

G-p

rote

in s

ignal

ing 2

, 24k

Da

9.0

E-0

9

-3

-3

C

ell

cycl

e, G

PC

R s

ignal

ing p

ath

way,

cell

com

mu

nic

atio

n

2580

5

BA

MB

I B

MP

an

d a

ctiv

in m

emb

ran

e-b

oun

d

inh

ibit

or

hom

olo

g (

Xen

op

us

laev

is)

9.7

E-1

0

-2

-3

T

GF

-β s

ignal

ing p

ath

way

Cel

lula

r m

ove

men

t, a

dh

esio

n,

inva

sio

n, cy

tosk

elet

on

rem

odel

ing

8979

7

NA

V2

N

euro

n n

avig

ator

2

6.7

E-0

9

9

10

2

9

Sca

ffold

pro

tein

, ac

tin

cyto

skel

eton

rem

od

elin

g,

ner

vou

s sy

stem

dev

elop

men

t [5

3]

2711

1

SD

CB

P2

S

yn

dec

an b

ind

ing p

rote

in 2

2

.2E

-03

6

1

5

B

ind

s S

DC

2,

regu

late

s ce

ll a

dh

esio

n a

nd c

om

mu

nic

atio

n w

ith

EC

M m

ole

cule

s [8

4, 85]

1134

4

PT

K9

L

Tw

infi

lin

9

.0E

-10

6

5

1

1

Act

in-b

indin

g p

rote

in, cy

tosk

elet

on

org

aniz

atio

n

[57]

8371

5

ES

PN

E

spin

2

.1E

-08

5

8

>

50

A

ctin

-bin

din

g-b

un

dli

ng p

rote

in, ac

tin c

yto

skel

eton

dyn

amic

s, c

ell

loco

moti

on

[5

4, 117

,

118]

5147

4

LIM

A1

E

pli

n,

LIM

dom

ain a

nd a

ctin

bin

din

g 1

3

.0E

-10

4

5

1

5

Cyto

skel

eton

-ass

oci

ated

pro

tein

, re

gula

tion

of

acti

n f

ilam

ent

dep

oly

mer

izat

ion

[5

6]

8829

N

RP

1

Neu

rop

ilin

1

4.1

E-0

8

4

11

4

0

TM

, co

rece

pto

r fo

r V

EG

F,

coll

agen

bin

din

g, ce

ll a

dh

esio

n, in

vas

ion

[1

19]

4582

M

UC

1

Muci

n 1

, ce

ll s

urf

ace

asso

ciat

ed

1.1

E-0

9

4

4

T

M,

cell

ad

hes

ion

, in

vas

ion

[1

20]

4131

M

AP

1B

M

icro

tubu

le-a

ssoci

ated

pro

tein

1B

7

.1E

-09

3

9

1

8

Tra

ns-

mem

bra

ne

gly

cop

rote

in,

cyto

skel

eton

org

aniz

atio

n

[55

, 121]

9644

S

H3

PX

D2

A

SH

3 a

nd

PX

dom

ain

s 2

A

1.4

E-0

9

3

7

A

dap

tor

pro

tein

, E

CM

deg

radat

ion

by a

ctin

bas

ed i

nvad

op

edia

form

atio

n

[122]

6275

S

100

A4

S

100

cal

ciu

m b

indin

g p

rote

in A

4

2.6

E-0

8

2

24

>

50

E

F-h

and c

alci

um

-bin

din

g p

rote

in,

pro

mote

s ce

ll m

oti

lity

, tu

mor

met

asta

sis

[49]

6383

S

DC

2

Syn

dec

an 2

3

.1E

-06

2

5

3

2

TM

, b

ind s

ign

alin

g f

acto

rs,

cell

adh

esio

n, an

gio

gen

esis

, E

CM

deg

radat

ion

[84

, 85]

1013

C

DH

15

C

adh

erin

15,

typ

e 1

, M

-cad

her

in

6.9

E-0

8

2

5

21

T

M,

Ca2

+d

epen

den

t in

terc

ellu

lar

adh

esio

n, sk

elet

al m

usc

le d

evel

op

men

t [1

23]

Sec

rete

d f

act

ors

1437

C

SF

2

Colo

ny s

tim

ula

tin

g f

acto

r 2

/GM

-CS

F

2.3

E-0

9

12

1

7

>50

C

yto

kin

e, o

steo

lyti

c b

on

e m

etas

tasi

s [7

2]

5225

P

GC

P

rogas

tric

sin

(p

epsi

nogen

C)

3.9

E-1

2

11

1

5

A

spar

tyl

pro

tein

ase

[47]

5304

P

IP

Pro

lact

in-i

ndu

ced

pro

tein

<

1.0

E-1

21

0

79

>

50

A

spar

tyl

pro

tein

ase,

lig

and

for

CD

4 r

ecep

tor

T c

ells

[46]

8857

F

CG

BP

F

c fr

agm

ent

of

IgG

bin

din

g p

rote

in

1.5

E-0

9

5

9

C

ell

adh

esio

n,

cell

-cel

l re

cogn

itio

n

6387

C

XC

L1

2

Ch

emokin

e l

igan

d 1

2 (

SD

F-1

) 2

.2E

-06

5

7

2

3

Ch

emokin

e, s

tem

cel

l tr

affi

ckin

g t

o b

on

e, m

igra

tion

, m

etas

tasi

s o

f tu

mor

cell

s

[58

-65]

7422

V

EG

FA

V

ascu

lar

end

oth

elia

l gro

wth

fac

tor

A

4.5

E-1

0

5

3

4

Gro

wth

fac

tor,

an

gio

gen

esis

, ce

llu

lar

morp

hogen

esis

du

rin

g d

iffe

ren

tiat

ion

[88

-91]

1907

E

DN

2

End

oth

elin

2

4.3

E-0

7

5

2

G

row

th f

acto

r, v

asoac

tive,

G-p

rote

in s

ign

alin

g,

smooth

mu

scle

contr

acti

on

[87]

5654

H

TR

A1

H

trA

ser

ine

pep

tid

ase

1

6.9

E-1

0

3

18

Ser

ine

pro

teas

e, d

iffe

ren

tial

ly e

xp

ress

ed i

n o

steo

arth

riti

c ca

rtil

lage

[124]

3817

K

LK

2

Kal

lik

rein

-rel

ated

pep

tid

ase

2

1.5

E-0

8

4

3

S

erin

e p

rote

ase,

pro

tein

met

aboli

c p

roce

ss

4880

N

PP

C

Nat

riu

reti

c p

epti

de

pre

curs

or

C

1.8

E-1

0

4

17

>

50

P

epti

de,

pote

nt

nat

riu

reti

c, d

iure

tic,

an

d v

asodil

atin

g

7057

T

HB

S1

T

hro

mb

osp

ond

in 1

3

.0E

-10

4

1

4

>50

C

ell

adh

esio

n, h

epar

in-b

indin

g, an

ti-a

ngio

gen

ic

3934

L

CN

2

Lip

oca

lin

2

5.1

E-0

8

4

10

5

E

CM

pro

tein

, p

rese

nt

ligan

ds

to c

ell

surf

ace

rece

pto

rs, p

rom

ote

s E

MT

[4

5]

6462

S

HB

G

Sex

horm

on

e-b

ind

ing g

lob

uli

n

8.7

E-1

0

4

10

Ste

roid

-bin

din

g,

sign

al

5764

2

CO

L2

0A

1

Coll

agen

, ty

pe

XX

, α

1

4.9

E-0

7

4

10

>

50

A

dh

esio

n,

esta

bli

shm

ent

of

loca

liza

tion

, tr

ansp

ort

4254

K

ITL

G

KIT

lig

and

2

.3E

-08

4

7

4

1

Hem

atop

oie

tic

gro

wth

fac

tor

and

lig

and f

or

the

KIT

tyro

sin

e k

inas

e re

cep

tor

4884

N

PT

X1

N

euro

nal

pen

trax

in I

2

.5E

-07

3

2

0

C

ell-

cell

sig

nal

ing, t

ran

sport

, est

abli

shm

ent

of

loca

liza

tion

3486

IG

FB

P3

IG

F b

indin

g p

rote

in 3

2

.2E

-08

3

7

Reg

ula

tion o

f ce

ll g

row

th,

com

munic

atio

n, ap

op

tosi

s

9806

S

PO

CK

2

Sp

arc/

ost

eon

ecti

n, cw

cv,

kaz

al-l

ike

dom

ain

s p

rote

ogly

can

(te

stic

an)

2

1.1

E-0

6

3

4

C

ell

dif

fere

nti

atio

n, E

CM

org

aniz

atio

n a

nd

bio

gen

esis

8530

C

ST

7

Cyst

atin

F (

leu

kocy

stat

in)

3.0

E-0

9

2

25

Com

pet

itiv

e in

hib

itors

of

cyst

ein

e p

rote

ases

, m

etas

tasi

s as

soci

ated

pro

tein

[4

8]

4885

N

PT

X2

N

euro

nal

pen

trax

in I

I 7

.6E

-09

2

6

Cel

l-ce

ll s

ign

alin

g,

syn

apti

c tr

ansm

issi

on

1473

81

CB

LN

2

AD

AM

MM

P w

ith

Thb

s-1 m

oti

f 5

.2E

-12

-3

-3

Pep

tid

ase,

cle

avag

e of

pre

cere

bel

lin

in

th

e n

ervou

s sy

stem

67

81

S

TC

1

Sp

ond

in 2

3

.6E

-08

-3

-3

EC

M p

rote

in, in

tegri

n l

igan

d, pat

tern

rec

ogn

itio

n

70

35

T

FP

I T

issu

e fa

ctor

pat

hw

ay i

nhib

itor

3.9

E-0

5

-3

-4

K

un

itz-

typ

e in

hib

itor

of

blo

od

coag

ula

tion

by b

lock

ing f

acto

r X

a

9518

G

DF

15

G

row

th d

iffe

ren

tiat

ion f

acto

r 15

1

.6E

-09

-2

-8

-7

G

row

th f

acto

r, T

GF

-β s

ignal

ing p

athw

ay,

cell

com

mu

nic

atio

n

Fo

ld c

han

ges

on

day

1 a

nd

day

2 a

re l

iste

d i

n c

olu

mn

s I

and

II

resp

ecti

vel

y,

and

colu

mn

q l

ists

fold

chan

ges

bas

ed o

n R

T-q

PC

R a

nal

ysi

s of

the

day

-2 s

amp

les.

Maj

or

gen

e fu

nct

ion

s w

ere

det

erm

ined

usi

ng k

now

led

ge

dat

abas

es f

rom

OM

IM (

On

lin

e M

end

elia

n I

nh

erit

ance

of

Man

), I

PA

™ s

yst

ems

and

th

e in

dic

ated

ref

eren

ced

lit

erat

ure

. A

bb

revia

tion

s: E

MT

, ep

ith

elia

l to

mes

ench

ym

al t

ransi

tion

; T

M,

tran

smem

bra

ne;

GP

CR

, G

-pro

tein

cou

ple

d r

ecep

tor;

EC

M, ex

trac

ellu

lar

mat

rix.

Table 2. Diseases, Molecular and Cellular Functions associated with genes up-regulated

(A) and down-regulated (B) by Runx2.

A) ≥ 2 fold up-regulated, total 532 genes p-value genes Diseases and disorders

Cancer 2.5e-07

- 3.0e-03 124

Genetic disorder 5.4e-07

- 3.0e-03 229

Inflammatory response 3.3e-06

- 2.4e-03 65

Molecular and cellular functions Cellular movement 9.7e

-12 - 2.5e

-03 84 Cell death 2.6e

-10 - 3.2e

-03 129 Cellular growth, proliferation 9.3e

-08 - 3.2e

-03 108 B) ≥ 2 fold down-regulated, total 378 genes

Diseases and disorders Cancer 3.3e

-29 - 4.1e

-03 124 Gastrointestinal disease 3.3e

-29 - 3.7e

-03 72 Genetic disorder 3.3e

-06 - 3.4e

-03 174 Molecular and cellular functions

Cell cycle 7.1e-35

- 4.1e-03 102

Cellular assembly, organization 1.7e-18

- 3.4e-03

63 DNA replication, recombination, repair 1.7e

-18 - 4.1e

-03 101

IPATM

analysis indicating the association of Runx2-regulated genes to a particular

category identifying the indicated disease or function. The ranges of p values indicate the

statistical significance with which Runx2-regulated genes associate with the each

category.

B

GAPDH

Runx2

Runx2-Mdox

0.1 0.5

Runx2dox

*c-Flag)

0.1 0.5--

A

C

0

100

200

300

400

500

Dox: *"og/ml)

6xOSE2-Luc

RLU

E

Runx2-MdoxRunx2dox

0.1 0.5- 0.1 0.5-

:Dox (og/ml)

D

F

Re

l. m

RN

A/G

AP

DH

0

0.1

0.2

0.3OC

Day: 1 2Day: 1 20

0.1

0.2

0.3MMP9

0

0.1

0.2

0.3

0.4BSP

*c-c-term)

Tubulin

-0.25

ROS 17/2.8

C4-2B/Rx2dox

Runx2

- :Dox (og/ml)

0.25-

Day 1 Day 2

Day: 1 2

QA

1 528

PSTRuntQA

1 528

PSTRunt

5’LTR FLAP TRE Ubi -c rtTA3 IRES Hyg WRE 3’SIN LTR5’LTR FLAP TRE Ubi -c rtTA3 IRES Hyg WRE 3’SIN LTR

constitutiveconstitutive

Dox inducedDox induced

Flag

Runx2:

R265D, R268D

pSLIK -Runx2

Re

l. m

RN

A/G

AP

DH

0

0.2

0.4

0.6

0.8

1.0

1.2Veh Dox

Day1 Day2

PC3highC4-2B/Rx2dox

Veh DoxVeh Dox

Figure 1

53

2 g

en

es in

du

ce

d œ

2-f

old

3

78

ge

ne

s r

ep

resse

d œ

2-f

old

Co

lor

co

de

pø

0.0

08

pø

0.0

08

Figure 2

Runx2

TGFd-RIII

Myc

PIP

SDF-1

VEGF

- + :Dox

ce

ll e

xtr

act

su

pern

ata

nt

CDK2

Tubulin

C4-2B/Rx2dox

Figure 3

250

150

100

75

50

- + :Dox

supernatant

C

+ D

ox

Ve

h

B

0

500

1000

1500

2000

No Matrigel™ Matrigel™

RLU

A

Veh Dox

Figure 4

Figure 5

Day 1

- +

Day 2

- +

E

Dox:

Ce

ll cycle

ph

ase

, p

erc

en

t

G2/MSG1

0

20

40

60

80

100

120

B

Via

bili

ty (

MT

T:

a.u

.x1

03)

0

1

2

3

4

5

6

7

0 2 4 6 8

Runx2dox

Veh

Dox

Day:

C

0

1

2

3

4

5

6

0 2 4 6 7

Runx2-Mdox

VehDox

Day:

Via

bili

ty (

MT

T:

a.u

.x1

03)

I

0

1

2

3

4

0 2 3 5 6

VehDox

Day

F

H

Day4 Day7Day10

0.2

0.8

1

1.2

1.4

Fo

ld c

asp

-3 a

ctivity

Runx2dox

Runx2-Mdox

1.6D

Plating-48 hr Day2 Day4 Day6

+ Dox

+ Veh

Ce

ll cycle

ph

ase

, p

erc

en

t

0

20

40

60

80

100

120

Dox: / -Day 2

*-+/Day 4

*-+/Day 6

Via

bili

ty (

MT

T:

a.u

.x1

03)

G

Runx2(c-flag)

GAPDH

Dox: / -day 2

*-+/day 4

*-+/day 6

0

0.001

0.002

0.003

0.004

n d

RASD1

0

0.02

0.04

0.06

0.08DUSP1

0

0.02

0.04

0.06 cMYC

0

0.002

0.004

0.006 E2F2

Re

l. m

RN

A/G

AP

DH

A Veh

Dox

Figure 5

Nucleus

Cytoplasm

Plasma Membrane

Extracellular Space

Figure 6

Additional files provided with this submission:

Additional file 1: Additional file 1.xls, 19906Khttp://www.molecular-cancer.com/imedia/1695821719450985/supp1.xlsAdditional file 2: Additional file 2.doc, 115Khttp://www.molecular-cancer.com/imedia/1643006969450986/supp2.docAdditional file 3: Additional file 3.xls, 79Khttp://www.molecular-cancer.com/imedia/1135951886450986/supp3.xlsAdditional file 4: Additional file 4.pdf, 21Khttp://www.molecular-cancer.com/imedia/7539305334509863/supp4.pdfAdditional file 5: Additional file 5.pdf, 48Khttp://www.molecular-cancer.com/imedia/1113604708450986/supp5.pdfAdditional file 6: Additional file 6.pdf, 28Khttp://www.molecular-cancer.com/imedia/1591460234509864/supp6.pdfAdditional file 7: Additional file 7.pdf, 19Khttp://www.molecular-cancer.com/imedia/7454516794509865/supp7.pdfAdditional file 8: Additional file 8.doc, 80Khttp://www.molecular-cancer.com/imedia/1301714918450986/supp8.docAdditional file 9: Additional file 9.doc, 188Khttp://www.molecular-cancer.com/imedia/1784406338450986/supp9.doc